Marino-Puertas et al. 1

Matrix metalloproteinases outside vertebrates

Laura Marino-Puertas, Theodoros Goulas* and F. Xavier Gomis-Rüth*

Proteolysis Lab; Structural Biology Unit; "María-de-Maeztu" Unit of Excellence; Molecular Biology Institute of Barcelona (CSIC); Barcelona Science Park; c/Baldiri Reixac, 15-21; 08028 Barcelona (Spain).

* Corresponding authors: e-mail: [email protected], phone: (+34) 934 020 187 or e-mail: [email protected], phone: (+34) 934 020 186.

ABSTRACT

The matrix metalloproteinase (MMP) family belongs to the metzincin clan of zinc-dependent metallopeptidases. Due to their enormous implications in physiology and disease, MMPs have mainly been studied in vertebrates. They are engaged in extracellular protein processing and degradation, and present extensive paralogy, with 23 forms in humans. One characteristic of MMPs is a ~165-residue catalytic domain (CD), which has been structurally studied for 14 MMPs from human, mouse, rat, pig and the oral-microbiome bacterium Tannerella forsythia. These studies revealed close overall coincidence and characteristic structural features, which distinguish MMPs from other metzincins and give rise to a sequence pattern for their identification. Here, we reviewed the literature available on MMPs outside vertebrates and performed database searches for potential MMP CDs in invertebrates, plants, fungi, viruses, protists, archaea and bacteria. These and previous results revealed that MMPs are widely present in several copies in Eumetazoa and higher plants (Tracheophyta), but have just token presence in eukaryotic algae. A few dozen sequences were found in Ascomycota (within fungi) and in double-stranded DNA viruses infecting invertebrates (within viruses). In contrast, a few hundred sequences were found in archaea and >1000 in bacteria, with several copies for some species. Most of the archaeal and bacterial phyla containing potential MMPs are present in human oral and gut microbiomes. Overall, MMP-like sequences are present across all kingdoms of life, but their asymmetric distribution contradicts the vertical descent model from a eubacterial or archaeal ancestor.

Keywords: zinc metalloproteinase; metzincin; MMP; invertebrates; catalytic domain; structure-based sequence motif

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1. Molecular characteristics of matrix metalloproteinases — The matrix metalloproteinases (MMPs) are a widespread family of zinc-dependent metallopeptidases (MPs), which either broadly degrade extracellular matrix components or selectively activate or inactivate other proteins through limited proteolysis [1-3]. MMPs were discovered in 1962 as an active principle in frog metamorphosis [4], and they contain a central zinc-dependent catalytic domain (CD) of ~165 residues, which is mostly furnished at its N-terminus with a ~20-residue signal peptide for secretion and an ~80-residue zymogenic pro-domain (PD). Some MMPs possess a "furin recognition motif" (R-X-R/K-R; [5]) for proteolytic activation between the PD and the CD. Into this minimal configuration, distinct MMPs have inserted extra segments and domains, such as fibronectin-type-II inserts within the CD and C- terminal unstructured linker regions, ~200-residue hemopexin domains (HDs) and other domains, as well as glycosyl phosphatidylinositol (GPI) anchors and transmembrane segments [1, 3, 6]. Overall, this variability divides human MMPs into subfamilies: archetypal MMPs, gelatinases, matrilysins and furin-activatable MMPs, which include membrane-type MMPs [6, 7]. MMPs belong to the metzincin clan of MPs, which currently comprises twelve structurally characterized families with common features [8-12]. However, this structural similarity is not reflected at sequence level, because Marino-Puertas et al. 2 pairwise identities are typically <20%, which is below the twilight zone of relevant sequence similarity (25–35%; [13]). Overall, metzincins share a globular CD of ~130—270 residues, which is split into a structurally conserved N-terminal sub-domain (NTS) and a diverging C-terminal sub-domain (CTS) by a horizontal active-site cleft. This cleft contains the catalytic zinc ion and accommodates protein or peptide substrates for catalysis (Figure 1A). NTSs contain a mostly five-stranded β-sheet, whose strands (βI-βV) parallel the active-site cleft. The lowermost strand βIV shapes the upper rim of the cleft and runs antiparallel to the other strands and to a bound substrate, thus establishing inter-main-chain interactions to fix it. NTSs also possess a "backing helix" (αA) with structural functions and an "active-site helix" (αB) with an extended "zinc-binding motif", H-E-X-X-H-X-X-G/N-X-X-H/D- Φ. This motif includes the general base/acid glutamate required for catalysis and three metal-binding protein residues (in bold), as well as a "family-specific residue" (Φ; [8-10]). Some families further comprise an additional "adamalysin helix", which was first identified in the adamalysin/ADAM family [12, 14]. In contrast to this structural conservation, CTSs largely deviate in length and structure but share the "Met-turn" [15], a loop with a methionine placed just below the catalytic zinc, and a "C-terminal helix" (αC) that terminates the CD. Into this common minimal scaffold, specific molecular elements decorate each metzincin family in the form of loops, ion- binding sites, additional regular secondary structure elements, extra domains, etc. [8-12]. To date, structural studies on MMP CDs are restricted to mammals (human, mouse, rat and pig), with the notable exception of bacterial karilysin ([12]; see also Section 6). These structures revealed that MMPs are very similar (Figure 1B), have much longer NTSs (~127 residues) than CTSs (~37 residues), and include an "S-shaped double loop", which connects the third and fourth strands of the NTS β-sheet (Figure 1A). This loop binds essential structural zinc and calcium cations, after which the polypeptide forms a prominent bulge ("bulge-edge segment") that protrudes into the active-site groove and assists in substrate specificity. A further hallmark of MMPs is a second calcium site within the NTS and that the family-specific residue is a serine or threonine [3, 10]. This residue makes a strong hydrogen bond between side chains with the first of two consecutive aspartates at the beginning of helix αC (Figure 1C). This aspartate is also engaged in fixing the N-terminus of the mature CD upon proteolytic activation. In turn, the second aspartate participates in a double electrostatic interaction with main-chain atoms of the Met-turn (Figure 1A,C). This intricate electrostatic network is structured around the "connecting segment", which links the third zinc-binding histidine with the Met-turn methionine and spans only seven residues; thus, it is the shortest of the metzincins [10]. Further typical of MMPs are two tyrosines, respectively four positions downstream of the Met-turn methionine and featuring the penultimate residue of the CD. Finally, the Met-turn and helix αC are connected by a loop subdivided into the "S1'-wall-forming segment" and the downstream "specificity loop" (Figure 1A). Taken together, all these characteristic structural features yield an extended sequence pattern, H- E-2X-H-2X-G-2X-H-S/T-6X-M-3X-Y-9X-D-D-7X-Y-X (Figure 1D), which distinguishes the C-terminal segment of MMP CDs from other metzincin families and MPs in general [12]. In addition to the CD, a further hallmark of MMPs is that latency is maintained through the PD [3], which together with transcriptional regulation and dedicated protein inhibitors regulates physiological MMP activity [16]. In most MMPs, zymogenicity is exerted by a "cysteine-switch" mechanism centered on a cysteine within a consensus motif at the end of the PD, P-R-C-G-X-P-D [17], which is found in all human MMPs except MMP-23B. Structurally, the polypeptide chain spanning these residues shields the active-site cleft by adopting a U-shaped conformation mediated by a double salt bridge between the arginine-aspartate pair and by the glycine, whose missing side chain prevents collision with upper-rim strand βIV [3]. In addition, the cysteine Sγ atom coordinates and thus blocks the catalytic zinc (see Fig. 3B in [3]). MMP activation entails PD removal to free access to the cleft. 2. Matrix metalloproteinases in invertebrates — MMPs have been extensively studied in vertebrates, particularly in mammals, due to their widespread implications in human health and disease [6, 16, 18, 19]. However, they are also widely present in invertebrates, where they participate in tissue turnover processes such as dendritic remodeling, regeneration, tracheal growth, axon guidance, histolysis and matrix degradation during hatching. They also participate in cancer processes [20]. Studied organisms belong to the phyla Arthropoda, including insects (dipterans, lepidopterans, hymenopterans and coleopterans) and crustaceans; Echinoderma, including sea urchins and sea cucumbers; Mollusca, including mussels and snails; Nematoda, Annelida and Planaria; invertebrate Chordata, including sea squirt and lancelet; and Cnidaria, including hydra and jellyfish (see Table 1). In addition, we found MMP-like sequences in the genomes of organisms as primitive as starlet sea anemone (Nematostella vectensis; UniProt database [www.uniprot.org] access code [UP] A7RJ22), which is also from the phylum Cnidaria, and sea gooseberry (Pleurobrachia pileus; GenBank database [www.ncbi.nlm.nih.gov/genbank] access code [GB] FQ005948) from the phylum Ctenophora, i.e. MMPs are present across the subkingdom Eumetazoa. In contrast, MMPs are apparently absent from the genomes of the sponge Amphimedon queenslandica (phylum Porifera; [21]) and of Trichoplax adherens (phylum Placozoa; [22]), which belong to the basalmost clades of metazoans.

Marino-Puertas et al. 3

Figure 1 — MMP catalytic domain structure. (A) Cross-eye stereographic Richardson-plot of the catalytic domain of T. forsythia karilysin (Y35-P200; PDB 2XS3; [23]) in standard orientation [24] as a representative of MMP catalytic domains (see [3]). Green arrows represent β-strands (labeled βI-βV), brown ribbons stand for α- helices (αA-αC). The two zinc cations found across MMPs are shown as magenta spheres, the calcium cations found in most MMPs but missing in karilysin are depicted in red. Relevant chain segments are shown in distinct colors and labeled (Met-turn in blue, specificity loop in red, S1'-wall- forming segment in yellow, S-loop in white, and bulge-edge segment in cyan). The side chains of the zinc-binding histidines, the general base/acid glutamate, the Met-turn methionine, the family-specific serine, and the two tyrosines and aspartates further included in the extended signature characteristic of MMPs are shown as stick models. (B) Superposition of the Cα-traces of the catalytic domains of bacterial karilysin (in yellow) and human MMP-1 (PDB 966C; [25]), MMP-3 (PDB 1CIZ; [26]), MMP-7 (PDB 1MMQ; [27]), MMP-8 (PDB 1JAN; [28]), MMP-9 (4H3X; [29]), MMP-10 (PDB 1Q3A; [30]), MMP-11 (PDB 1HV5; [31]), MMP-12 (PDB 1Y93; [32]), MMP-13 (PDB 2D1N; [33]) and MMP-16 (PDB 1RM8; [34]), all in cyan. The two consensus zinc and calcium cations are shown as magenta and red spheres, respectively; the consensus N- and C- termini are indicated by red arrows. (C) Close-up view of (A) highlighting the structural features of the C-terminal sub- domain characteristic for MMPs. The hallmark electrostatic interactions within the CTS are shown as cyan lines, residues included in the extended sequence pattern are depicted for their side chains and labeled. (D) Color-coded mapping of the MMP extended sequence pattern (H-E-2X- H-2X-G-2X-H-S/T-6X-M-3X-Y-9X-D-D- 7X-Y-X) onto the karilysin polypeptide chain. Each residue type is shown in one color and labeled, random residues (X) are alternatively in light green and yellow.

Eumetazoans are animals comprised of tissues organized into germ layers, with a gastrula stage during embryogenesis. Primitive animals of this lineage up to the emergence of the clade Bilateria likely had few MMP copies within their genomes, as currently found in several Drosophila species (two copies) and other insects such as red flour beetle, silkworm or malaria mosquito (three copies) [6, 20, 35-38]. In contrast, higher animals show gene polyplication, which likely arose during early vertebrate evolution and led to the substantial paralogy that is currently found, e.g., in humans (24 genes), mice (23 genes) and zebrafish (26 genes). However, development of this complexity was not linear: while seven genes are found in the sea squirt Ciona intestinalis and nine in the mosquito Aedes aegypti, the sea urchin Strongylocentrotus purpuratus has 26 [6, 20, 39-44].

Marino-Puertas et al. 4

Table 1 — MMPs reported from invertebrates

Phylum Annelida Class Polychaeta Boneworm (Osedax japonicus) [45]

Phylum Arthropoda Class Branchiopoda Water flea (Daphnia pulex) [46] Class Insecta Yellow-fever mosquito (Aedes aegypti) [44] Malaria mosquito (Anopheles gambiae) [47] Honeybee (Apis mellifera) [48] Silkworm (Bombyx mori) [49] Fruitfly (Drosophila melanogaster) [20, 35, 36]) Greater wax moth (Galleria mellonella) [50] Tobacco hornworm (Manduca sexta) [51] Red flour beetle (Tribolium castaneum) [37] Cabbage looper (Trichoplusia ni) [52]

Phylum Chordata Class Ascidiacea Sea squirt (Ciona intestinalis) [41] Class Leptocardii Lancelet (Branchiostoma japonicum) [53]

Phylum Cnidaria Class Hydrozoa Common hydra (Hydra vulgaris / Hydra magnipapillata) [54, 55] Class Scyphozoa Nomura's jellyfish (Nemopilema nomurai) [56]

Phylum Echinodermata Class Echinoidea Asian sea urchin (Hemicentrotus pulcherrimus) [57] Mediterranean purple sea urchin (Paracentrotus lividus) [58] Pacific purple sea urchin (Strongylocentrotus purpuratus) [40] Class Holothuroidea Japanese sea cucumber (Apostichopus japonicus) [59] Rock sea cucumber (Holothuria glaberrima) [60]

Phylum Mollusca Class Bivalvia American oyster (Crassostrea virginica) [61] Mediterranean mussel (Mytilus galloprovincialis) [62] Class Gasteropoda Giant African snail (Achatina fulica) [63] Bloodfluke planorb (Biomphalaria glabrata) [64] Many-colored abalone (Haliotis diversicolor) [65] Red abalone (Haliotis rufescens) [66] Korean common dogwhelk (Thais clavigera) [67]

Phylum Nematoda Class Secernentea Rat lungworm (Angiostrongylus cantonensis) [68] Roundworm (Caenorhabditis elegans) [69-71] Yellow potato cyst nematode (Globodera rostochiensis) [72] Parasitic nematode (Gnathostoma spinigerum) [73] Soybean cyst nematode (Heterodera glycines) [72]

Phylum Platyhelminthes Class Rhabditophora Freshwater planarian flatworm (Dugesia japonica) [74] Freshwater planarian flatworm (Schmidtea mediterranea) [74]

Taxonomy according to the Catalogue of Life (http://www.catalogueoflife.org/col; [75]).

Marino-Puertas et al. 5

The complex evolution of MMPs in Eumetazoa is also reflected by the presence of several ancillary domains in the full-length enzymes (see Section 1 and Fig. 1 in [6]). It can be speculated that primitive MMPs consisted of standalone CDs or possibly PD+CD tandems, which underwent duplication, gene fusion and exon shuffling to result in multi-domain architectures [6, 39, 76, 77]. However, in some instances evolution progressed in the opposite direction, i.e. multi-domain enzymes underwent truncation to yield proteins of fewer domains, even in mammals. This is the case for matrilysins, which span only a signal peptide, a PD with a cysteine-switch motif and a CD [6, 78, 79]. This minimal architecture is also predominant across invertebrates [6], although many MMP sequences in insects—e.g. in Drosophila [20]—further comprise a furin recognition sequence, an HD and a GPI anchor.

Table 2 — MMPs referenced from plants and selected sequences within genomes

Phylum Tracheophyta Class Liliopsida Barley (Hordeum vulgare): F2DC11, F2D2W1, F2DGF5, F2D593 [81] Rice (Oryza sativa): A2X9G3, A2YB49, A2ZA53, A2ZA54, A2ZA55, A2ZA56, [81] A2ZA53, A2ZA53 Sugar cane (Saccharum hybrid cultivar): A0A059Q041 [82] Corn (Zea mays): B6U4D1, B4FZU1 [81] Eelgrass (Zostera marina): A0A0K9P9J4*, A0A0K9PG73*, A0A0K9PXQ4*, A0A0K9PK10*, A0A0K9PLB4*, A0A0K9PY38*, A0A0K9PBG9*, A0A0K9P805* Class Magnoliopsida Lyre-leaved rock cress (Arabidopsis lyrata): D7LBU9* Mouse-ear cress (Arabidopsis thaliana): At1/2/3/4/5-MMP (O23507, O04529, [83-88] Q5XF51, Q8GWW6, Q9ZUJ5) Sugar beet (Beta vulgaris): A0A0J8F9H7, A0A0J8B8R0, GB XP_010692448, GB [89] XP_010695895 Cucumber (Cucumis sativus): Cs1-MMP (Q9LEL9) [90] Soybean (Glycine max): Gm1/2-MMP, Gm-Slti114-MMP (C6TNN5, Q93Z89, [81, 91-96] B6CAM2) and C6TNN5 Tree cotton (Gossypium arboreum): A0A0B0ME77* Barrel medic (Medicago truncatula): Mt1-MMP (Q9ZR44) [97] Tobacco (Nicotiana tabacum): Nt1-MMP (C3PTL6) [98, 99] Western balsam poplar (Populus trichocarpa): B9I3X8* Castor bean (Ricinus communis): B9RUG7* Tomato (Solanum lycopersicum): Sl1/2/3/4/5-MMP (I7JCM3, I7KJ40, K4BWG3, [81] K4CNL4, A0A0G3ZAU2, K4CYZ6) Cocoa (Theobroma cacao): A0A061DVV2*, S1S448* Class Pinopsida Loblolly pine (Pinus taeda): Pta1-MMP (B7TVN4) [100]

All sequence codes are from UniProt (UP; www.uniprot.org) or GenBank (GB; www.ncbi.nlm.nih.gov/genbank). Taxonomy according to the Catalogue of Life (http://www.catalogueoflife.org/col; [75]). Nomenclature of validated plant MMPs according to [101]. * Sequences annotated in UniProt as "matrix metalloproteinases" and manually curated. Searches completed on 10 January, 2017.

3. Matrix metalloproteinases in plants and algae — In addition to Eumetazoa, MMPs have been reported from higher plants (phylum Tracheophyta), where they are generally present in lower copy numbers than in animals. Enzymes were described from soybean, mouse-ear cress, cucumber, barrel medic, tobacco, loblolly pine and tomato (see Table 2). In addition, sequences were referenced from sugar beet, rice, corn, sugar cane and barley (Table 2). Moreover, MP activity attributable to an MMP was also described for the jack bean Canavalia ensiformis, though validation is still pending [80]. Finally, current sequence similarity searches identified several hundreds of potential hits in higher plants, Table 2 provides a curated selection of them. Plant MMPs localize to the plasma membrane or the extracellular space and have been found to be involved in remodeling of the extracellular matrix during plant growth and development processes, such as germination, programmed cell death and senescence, as well as in biotic and abiotic stress responses [86, 101-103]. Sequence analyses revealed that plant MMPs have a homogenous domain distribution and mostly comprise a signal peptide, a PD with a cysteine-switch motif that occasionally diverges from the consensus (see Section 1 and [102]), and a CD. Within the latter, some sequences have the general base/acid glutamate mutated to glutamine [102], a change that in mammalian MMPs leads to ablation or strong reduction of proteolytic activity [104]. Uniquely, plant MMPs Marino-Puertas et al. 6 encompass a specific consensus sequence, D-L-E-S/T, two residues upstream of the zinc-binding motif [81, 84, 101]. In addition, the loop that connects strand βV with helix αB is up to 10 residues longer in plant MMPs than in mammalian counterparts. In the absence of experimental structures, these two combined features point to a specific structural element of plant MMPs, putatively an extra cation-binding site. Downstream of the CD, plant MMPs only contain ~40-residue C-terminal GPI anchors or transmembrane segments for localization to the plasma membrane [81]. The unicellular green alga Chlamydomonas reinhardtii encodes two MPs dubbed gametolysins (also known as gamete lytic enzymes, MMP1 and MMP2, and autolysins), which are engaged in cell-wall turnover and have been recurrently associated with the MMP family [101, 102, 105-108]. Similarly to MMPs, these ~635-residue enzymes comprise a signal peptide and a putative PD with a cysteine-switch-like motif. These enzymes are similar to four enzymes from the multicellular green alga Volvox carteri (VMP1-VMP4), in which the first zinc-binding histidine is replaced with glutamine [109-111]. Current sequence similarity searches identified new potential paralogues of these MPs in C. reinhardtii and V. carteri, as well as in the green algae Gonium pectorale and Chlorella variabilis. However, in the absence of three-dimensional structures, the primary sequences of these MPs deviate from the extended sequence pattern of MMPs (see Section 1). Consequently, they belong to a separate metzincin family, which was tentatively dubbed gametolysins in the past [10, 12, 108]. This is consistent with their adscription to a family separate from MMPs within the MEROPS peptidase database (M11 vs. M10; see merops.sanger.ac.uk; [112]). Of note, searches for true MMPs within eukaryotic algae revealed six potential relatives, two in phytoplankton Emiliania huxleyi (UP R1DV14 and R1E2E0/R1EHE7) and one each in G. pectorale (UP A0A150GPR5), Symbiodinium minutum (KEGG Gene symbB.v1.2.029330.t2; see www.genome.jp), Aureococcus anophagefferens (UP F0Y382) and V. carteri (UP D8UD00). This restricted presence of MMPs within algae is consistent with recent genome-wide analyses of the secretomes of nine brown algae belonging to the phyllum Phaeophyceae, which revealed no potential MMP ortholog [113]. Furthermore, no other sequences were presently found in other Protista/Chromista. 4. Matrix metalloproteinases in fungi — Although some MPs were described from fungi [114-116], none has yet been confirmed to be an MMP. We thus performed a database search, which revealed several potential sequences in the phylum Ascomycota but not in other phyla (Table 3). These hypothetical proteins span between 255 and 659 residues, some have their catalytic glutamate replaced with glutamine (see Sections 1 and 3), and most comprise N-terminal extensions with a potential cysteine-switch-like motif. Those lacking this motif possess potential NTSs that are significantly shorter than in standard MMPs, so they may correspond to incomplete sequences or truncated variants. Several sequences also contain large N- and/or C-terminal extensions that could correspond to additional domains. Hit fungal species contain only one sequence each, with the exception of Arthrobotrys oligospora and Dactylellina haptotyla, with five sequences each, and all but two species are fungal pathogens of higher plants, nematodes, insects and animals—including humans—or endophytic fungi. Accordingly, their lifestyle entails very intimate contact with organisms that contain MMPs. 5. Matrix metalloproteinases in viruses — In 2000, the functional characterization of an MMP from Xestia c- nigrum granulovirus was reported [117]. In the absence of structural information, analysis of its amino acid sequence reveals an MMP CD. The upstream N-terminal segment lacks the canonical cysteine-switch motif, but shows sequence stretch C42-G-G-G-N-H-R-R-T-K-R52 immediately before the predicted mature N-terminus, which includes a cysteine-glycine pair reminiscent of those motifs, as well as a recognition sequence characteristic of furin-activatable MMPs (see [6] and Section 1). This notwithstanding, the full-length protein was active, thus suggesting it was in an at least partially competent state without hypothetical activation [117]. Threading calculations (see the legend to Figure 2A) with the protein segment downstream of the CD of Xestia MMP indicated that it most likely contained an HD (Figure 2A), preceded by an intermediate "threonine-rich region". More recently, Cydia pomonella granulovirus was also shown to express a functional MMP, the only other viral family member studied to date [38]. Like the Xestia ortholog, it contained a CD preceded by a potential furin- recognition sequence. However, it lacked any cysteine in the N-terminal fragment, so its function and/or activation mechanism might diverge from Xestia MMP. In contrast to previous hypotheses [38] but in agreement with the prediction for Xestia MMP, threading calculations indicated that a threonine-rich region and an HD are present in the C-terminal part of the protein (Figure 2B). Granuloviruses belong to the genus Betabaculovirus within the family Baculoviridae and all species sequenced to date within this genus contain putative MMP homologs (see Suppl. Table 1 and [38]). In contrast, MMPs are absent from the other Baculoviridae geni, viz. Alphabaculovirus, Deltabaculovirus and Gammabaculovirus. In addition to Baculoviridae, we also found MMP sequences in the families Iridoviridae, Nudiviridae, Poxviridae (subfamily Entomopoxvirinae) and Ascoviridae (Suppl. Table 1) but not in any other viruses or viroids. Collectively, all these virus families are double-stranded DNA viruses with no RNA stage, which infect invertebrates (arthropods, lepidopters, hymenopters, diptera and decapods), i.e. organisms that contain MMPs. The potential viral MMPs vary in length, some contain a signal peptide, a potential cysteine-switch-like motif, a Marino-Puertas et al. 7 putative furin-cleavage site, a predicted HD and a threonine-rich region as in Xestia and Cydia MMPs, but others do not.

Table 3 — Fungal MMP sequences

Phylum Ascomycota Class Dothideomycetes Bipolaris oryzae 388 residues GB XP_0077684757 / UP W6ZMI0 Bipolaris victoriae 388 residues GB XP_014556326 / UP W7EIS0 Bipolaris zeicola 388 residues GB XP_007709037 / UP W6YG21 Cochliobulus heterostrophus 388 residues GB XP_014075121 / UP N4X7I4, UP (Bipolaris maydis) M2V381 ** Cochliobulus sativus 320 residues GB XP_007696738 / UP M2TCL8 (Bipolaris sorokiniana) Paraphaeosphaeria sporulosa 411 residues GB XP_018039150 / UP A0A177CMM7 Phaeosphaeria nodorum 326 residues GB XP_001794978 / UP Q0UUK1 (Parastagonospora nodorum) (Septoria nodorum) Pyrenochaeta sp. 565 residues UP A0A178DJW7 Stagonospora sp. 393 residues UP A0A178BDB5 Stemphylium lycopersici 563 residues UP A0A0L1HKC2 Class Eurotiomycetes Aspergillus calidoustus 290 residues UP A0A0U5G9D2 Aspergillus flavus 274 residues GB XP_002379978 / UP B8NI13, UP A0A0D9MRC7 ** Aspergillus lentulus 311 residues UP A0A0S7DKQ5 Aspergillus udagawae 282 residues UP A0A0K8L198 Endocarpon pusillum 306 residues GB XP_007803399 / UP U1HKZ8 Exophiala aquamarina 282 residues GB XP_013264742 / UP A0A072PQX4 Neosartorya fischeri (Aspergillus 616 residues GB XP_001261124 / UP A1DIM0 fischerianus) Class Leotiomycetes Pseudogymnoascus sp. 255 residues UP A0A094DQI1, UP A0A094ITE6 * Class Orbiliomycetes Arthrobotrys oligospora 609 residues GB XP_011127847 / UP G1XU64 Arthrobotrys oligospora 599 residues GB XP_011127846 / UP G1XU63 Arthrobotrys oligospora 290 residues GB XP_011126672/ UP G1XQB0 Arthrobotrys oligospora 315 residues GB XP_011126207 / UP G1XNZ5 Arthrobotrys oligospora 232 residues GB XP_011120912 / UP G1X8V0 Drechslerella stenobrocha 210 residues UP W7IFU4 Class Sordariomycetes Dactylellina haptotyla 659 residues GB XP_011116834 / UP S7ZXB5 Dactylellina haptotyla 308 residues GB XP_011114406 / UP S8A464 Dactylellina haptotyla 292 residues GB XP_011112095 / UP S8AFF5 Dactylellina haptotyla 268 residues GB XP_011113422 / UP S8BTL0 Dactylellina haptotyla 246 residues GB XP_011107496 / UP S8AU63 Metarhizium anisopliae 619 residues UP A0A0B4G9D2 Metarhizium anisopliae 607 residues UP A0A0D9NZH8 Metarhizium brunneum 619 residues GB XP_014544223 / UP A0A0B4FXN2 Metarhizium robertsii 619 residues GB XP_007825075 / UP E9F9D7, A0A014N9M3 ** Pochonia chlamydosporia 636 residues GB XP_018148437 / UP A0A179G3B6 Purpureocillium lilacinum 627/629 residues GB XP_018174930 / UP A0A179GT13, A0A179GDA3 *

All sequence codes are from UniProt (UP; www.uniprot.org) or GenBank (GB; www.ncbi.nlm.nih.gov/genbank). Sequence searches were performed with structurally validated MMP CDs within UniProt (www.uniprot.org) or the National Center for Biotechnology Information (blast.ncbi.nlm.nih.gov/Blast.cgi) using standard parameters. Taxonomy according to the Catalogue of Life (http://www.catalogueoflife.org/col; [75]). * These sequences have only minimal differences. ** These entries are identical. Searches completed on 19 January, 2017.

Marino-Puertas et al. 8

Moreover and consistent with the host specificity of the harboring viruses, sequences cluster closely with insect MMPs [38], which possess a similar domain architecture (see Section 2 and [20]). In insects, housekeeping MMPs participate in physiological remodeling of the basal lamina, which lines the midgut to prevent systemic infections [126]. In turn, as part of the infective process, ingested viral particles reach the midgut of target insects and breach the basal lamina, a task that might be carried out by viral MMPs [126].

Figure 2 — Predicted structure of baculoviral MMP C-terminal domains. (A) Superposition in cross-eye stereo of the two top-ranked homology models (in tan and cyan respectively) of Xestia c- nigrum granulovirus MMP segment C280-C469 (UP Q9PZ03) by threading calculations with the LOMETS meta-server using programs cdPPAS (Z- score 16.9; [118]) and SP3 (Z-score 36.6; [119]), respectively. Full-length modeling was automatically carried out with the program MODELLER [120]. The corresponding Cα-traces and the critical disulfide bond linking the terminal β- leaflets (C280-C469; red arrow), which is usually found in fourfold β-propeller structures such as MMP hemopexin domains [121], are depicted. In both cases, the automatically selected template structure was that of the hemopexin domain of human MMP-14 (PDB 3C7X; [122]). (B) Same as (A) but for Cydia pomonella granulovirus MMP (UP Q91F09) segment C359-C545. The two top predictions (in purple and green, respectively) were obtained with the program HHSEARCH (Z-scores 29.6 and 35.9; [123]). Selected templates were the hemopexin domains of human MMP-2 (PDB 1GEN; [124]) and MMP-1 (PDB 1SU3; [125]). In the second automatic homology model, the two cysteines are not linked but close to each other in space.

6. Matrix metalloproteinases in archaea and bacteria — The only prokaryotic MMP characterized at the functional and structural level to date is karilysin from Tannerella forsythia. This is a human oral-microbiome bacterium from the phylum Bacteroidetes that is engaged in odontopathogenic infections [23, 127-135]. Karilysin comprises a CD flanked upstream by a signal peptide for secretion and a 14-residue PD, which does not proceed over a cysteine-switch mechanism but an "aspartate-switch" mechanism, as found in the otherwise unrelated astacin family within the metzincin clan [23, 128, 136, 137]. The CD is followed by two domains of unknown structure and function, collectively spanning 275 residues, which comprise the C-terminal residues K-L-I-K-K. A C-terminal domain similar to karilysin is found in other unrelated peptidases within T. forsythia, Bacteroides sp. and Prevotella sp., which have been collectively termed KLIKK proteases [138] The CD of karilysin was characterized at the structural level (see Figure 1A), which revealed that it fulfilled all the structural criteria of MMP CDs of mammals [23] (Figure 1B). These studies also suggested that karilysin is evolutionary closer to forms from mosquitoes that are insect vectors of malaria (Anopheles gambiae), dengue fever, Chikungunya and yellow fever (Aedes aegypti), and West Nile virus and Zika virus infections (Culex quinquefasciatus) than to bacterial counterparts [23]. The lifecycle of these mosquitoes entails feeding on human blood and they are mostly found in poor countries, which have the highest incidence of odontopathogenic bacterial infections [139]. Further to karilysin in bacteria, a potential MMP ortholog in Bacillus anthracis, MmpZ, was shown to participate in the extracellular degradation of anthrax toxin components and anthrolysin O at the onset of the stationary growth phase of the bacterium [140]. However, detailed inspection of its protein sequence (UP Q81NM7) reveals that it deviates from the extended sequence pattern of MMP CTSs. Therefore, it cannot be assigned unambiguously to MMPs until its three-dimensional structure has been resolved. Moreover, there have been other reports postulating the existence of bacterial MPs, which were hailed as ancestral forms of MMPs [43]. In particular, Bacteroides fragilis toxins alias fragilysins were thought to accomplish this role [39, 141-143]. However, when the structures of profragilysin-3 and the closely-related metalloproteinase II were reported, it became obvious that fragilysins, which are present in enterotoxigenic B. fragilis strains but not commensal ones, represent a metzincin family on their own, which is closer to adamalysin/ADAMs than MMPs, if at all [12, 144- 146]. Marino-Puertas et al. 9

To complete the picture of MMP distribution in prokaryotes, we conducted sequence similarity searches and identified several hundred potential MMP orthologs across archaeal and bacterial genomes, some of them with several copies. A representative selection of manually curated sequences is provided in Suppl. Tables 2 and 3. Inspection of the archaeal sequences reveals that they cluster to phyla Euryarchaeota and Thaumarchaeota, which populate the human digestive tract together with Crenarchaeota [147]. The healthy gut microbiome is also dominated in humans by the bacterial phyla Firmicutes and Bacteroidetes, with Actinobacteria, Proteobacteria and Verrucomicrobia present in smaller proportions [148]. Upstream in the gastrointestinal tract, six major bacterial phyla populate the oral microbiome: Firmicutes, Bacteroidetes, Proteobacteria, Actinobacteria, Spirochaetes, and Fusobacteria [149]. Bacterial species that potentially contain MMPs also belong to these phyla, as does karilysin, with the notable exception of a few bacterial sequences from the phyla Planctomycetes, an aquatic phylum present in brackish and fresh marine waters, and Cyanobacteria, which also inhabit waters and moist soils (Suppl. Table 3). Accordingly, the distribution of archaeal and bacterial sequences is patchy and they are almost exclusively found in species of phyla highly represented in human microbiomes. 7. Conclusion — MMPs are arguably the best studied MPs at the molecular, functional, physiological and structural levels, but most reports are restricted to humans and a few other animals. However, a comprehensive review of the literature and current sequence similarity searches revealed that validated and potential MMP CDs are widespread. In general, most proteins possess common ancestors in Eubacteria or Archaea, so their presence within the latter indicates that inheritance follows the Darwinian tree-based pathway or vertical descent model [77, 150]. This is the case for ~60% of human protein domains, which have their origins in these kingdoms and eukaryotic nodes before the metazoan era [77]. However, some proteins originate at nodes that appeared later in evolution [151], as reported for the large, multi-domain pan-peptidase inhibitors of the α2-macroglobulin (α2M) family [152]. These >1,000-residue proteins are widely distributed across metazoans, but missing in all non- metazoan eukaryotic lineages. Unexpectedly, homologous proteins were found in several bacterial proteomes [153, 154], but their distribution was patchy and incompatible with vertical descent from a common ancestral eubacterium. As most of these bacterial species encoding α2Ms exploited higher eukaryotes as hosts, either as pathogenic invaders or commensal colonizers, it was proposed that they were acquired by eukaryotic-to- prokaryotic horizontal gene transfer [152], similarly to previously suggested for some metabolic enzymes [155, 156]. MMPs are likewise widespread, perhaps across all kingdoms of life, where they are possibly involved in extracellular processing of proteins. However, they only show a homogenous gene distribution that is probably consistent with a vertical descent model within animals of the subkingdom Eumetazoa, as they are absent from more primitive metazoans. Within plants, they have only been found in higher plants. Here, the domain architecture is reminiscent of invertebrate MMPs, which suggests that plant and invertebrate MMPs are more closely related to each other than to vertebrate MMPs. This, in turn, hints that they could be modern representatives of an ancient MMP ancestor, common to the three groups [6, 39]. In fungi, protists, viruses, bacteria and archaea, the presence of MMP sequences is reduced and patchy, which violates the vertical descent model. Generally, distribution is restricted to phyla with a lifecycle entailing intimate, direct or indirect, pathogenic or commensal, interaction with members of the subkingdom Eumetazoa. This suggests that MMPs in those kingdoms, like bacterial α2Ms, are xenologs coopted several times during evolution from eumetazoan hosts by independent horizontal gene transfer events (see e.g. Suppl. Fig. 1), which would include uptake of mRNA by competence or abiotic mechanisms [157], followed by subsequent spreading and polyplication within phyla.

ACKNOWLEDGMENTS

This study was supported in part by grants from European, Spanish, and Catalan agencies (grant references FP7-HEALTH-2012-306029-2 “TRIGGER”; BFU2015-64487R, MDM-2014-0435; BIO2013-49320-EXP; BIO2015-64216-P; BIO2013-49604-EXP; and 2014SGR9). The Structural Biology Unit (www.sbu.csic.es) of IBMB is a “María de Maeztu” Unit of Excellence from the Spanish Ministry of Economy, Industry and Competitiveness.

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Marino-Puertas et al. 1

Supplementary Information of Matrix metalloproteinases outside vertebrates

Laura Marino-Puertas, Theodoros Goulas and F. Xavier Gomis-Rüth

Marino-Puertas et al. 2

Supplementary Figures

Suppl. Fig. 1: Phylogenetic studies. (A) Rooted phylogenetic tree reflecting evolutionary distances among selected protein sequences (in parenthesis the UniProt codes) of organisms from different kingdoms of life. Names in red are mammals, in green plants, in orange fungus, in lilac viruses, in blue bacteria and in black archaea. In blue background are highlighted proteins of different kingdoms (bacterial and fungal) that display high sequence similarity (see in B). This could be an indication of an eukaryotic-to-prokaryotic horizontal gene transfer event. The bar represents 0.2 substitutions per site. (B) Alignment of protein sequences from Arthrobacter nitrophenolicus (UniProt code: L8TT82) and Purpureocillium lilacinum (UniProt code: A0A179GT13) displaying more than 42% sequence identity in the blue backgrounded area. In red rectangle the zinc-binding motif (H-E-X- X-H-X-X-G/N-X-X-H/D) found in MMPs.

Marino-Puertas et al. 3

Supplementary Tables

Suppl. Table 1 — Viral MMP sequences

Family Ascoviridae Genus Ascovirus Heliothis virescens ascovirus 3e 474 residues GB YP_001110872 / UP A4KX75 Heliothis virescens ascovirus 3f 439 residues GB AJP08985 / UP A0A171PVB2 Heliothis virescens ascovirus 3g 439 residues GB AFV50272 / UP K4NY21 Spodoptera frugiperda ascovirus 1a 386 residues GB YP_762369 / UP Q0E587 Trichoplusia ni ascovirus 2c 501 residues GB YP_803380 / UP Q06VC5 Genus Toursvirus Diadromus pulchellus ascovirus 4a 223 residues GB YP_009220674 /UP F2NYV8

Family Baculoviridae Genus Betabaculovirus Adoxophyes orana granulovirus 395 residues GB NP_872491 / UP Q7T9X8 Cryptophlebia leucotreta granulovirus 486 residues GB NP_891890 / UP Q7T5P6 Cydia pomonella granulovirus # 546 /545 GB AIU36692 / UP A0A097P0M6 // GB NP_148830 / UP residues Q91F09 * Helicoverpa armigera granulovirus 596 residues GB YP_001649020 / UP A9YMN0 Phthorimaea operculella granulovirus 469 residues GB NP_663206 / UP Q8JS18 Plodia interpunctella granulovirus 620 residues GB YP_009330170 / UP Plutella xylostella granulovirus 402 / 403 GB NP_068254 / UP Q9DVZ7 // GB ANY57554 / UP residues A0A1B2CSG4 * Pseudalatia unipuncta granulovirus 593 residues GB YP_003422377 / UP B6S6Q7 Trichoplusia ni granulovirus LBIV-12 593 residues GB AOW41373 / UP A0A1D8QL47 Xestia c-nigrum granulovirus # 469 residues GB NP_059188 / UP Q9PZ03 Unclassified Betabaculoviridae Agrotis segetum granulovirus 481 residues GB YP_006303 / UP Q6QXG0 Choristoneura occidentalis granulovirus 488 residues GB YP_654454 / UP Q1A4R3 Clostera anachoreta granulovirus 412 residues GB YP_004376233 / UP F4ZKQ2 Clostera anastomosis granulovirus 412 residues GB YP_008719974 / UP U5KB82 Clostera anastomosis granulovirus 486 residues GB AKS25377 / UP A0A0K0WSE3 Cnaphalocrocis medinalis granulovirus 441 residues GB ALN41975 / UP A0A0X9FQ45 // GB YP_009229958 / UP A0A109WW48 * Diatraea saccharalis granulovirus 370 residues GB YP_009182238 / UP A0A0R7EZ65 Epinotia aporema granulovirus 359 residues GB YP_006908552 / UP K4ERV0 Erinnyis ello granulovirus 462 residues GB YP_009091878 / UP A0A097DAI6 Mocis sp. granulovirus 580 residues GB YP_009249873 / UP A0A162GWM9 Pieris rapae granulovirus 432 residues GB YP_003429361 / UP D2J4K4 Pieris rapae granulovirus 444 residues GB ADO85463 / UP E7BN22 Spodoptera frugiperda granulovirus 539 residues GB YP_009121819 / UP A0A0C5B309 Spodoptera litura granulovirus 464 residues GB YP_001256988 / UP A5IZN9

Family Iridoviridae Genus Iridovirus Invertebrate iridescent virus 6 (Chilo 264 residues GB NP_149628 / UP O55761 iridescent virus) Invertebrate iridovirus 22 352 residues GB YP_008357315 / UP S6DDP6 Invertebrate iridescent virus 22 365 residues GB YP_009010779 / UP W8W1A0 Invertebrate iridovirus 25 362 residues GB YP_009010550 / UP W8W2D9 Invertebrate iridescent virus 30 367 residues GB YP_009010310 / UP W8W249 Wiseana iridescent virus (Insect 346 residues GB YP_004732919 / UP G0T5G2 iridescent virus type 9) Genus Chloriridovirus Aedes taeniorhyncus iridescent virus 363 residues ** GB ABF82125 Invertebrate iridescent virus 3 (IIV-3) 363 residues ** GB YP_654667 / UP Q196W5 Marino-Puertas et al. 4

(Mosquito iridescent virus) Unclassified Iridoviridae Anopheles minimus irodovirus 366 residues GB YP_009021109 / UP W8QE20

Family Nudiviridae Genus Betanudivirus Helicoverpa zea nudivirus 2 (HzNV-2) 789 residues GB YP_004956816 / UP G9I094 Heliothis zea nudivirus 792 residues GB AAN04364 / UP Q8JKP2

Family Poxviridae (Subfamily Entomopoxvirinae) Genus Alphaentomopoxvirus Anomala cuprea entomopoxvirus 268 residues GB YP_009001641 / UP W6JIV8 Genus Betaentomopoxvirus Amsacta moorei entomopoxvirus 252 residues GB AAG02776, GB NP_064852 / UP Q9EMX9 ** (AmEPV) Mythimna separata entomopoxvirus 'L' 411 residues GB YP_008003705 / UP R4ZFL1

Unclassified dsDNA viruses, no RNA stage Apis mellifera filamentous virus 830 residues GB AKY03287, GB YP_009165969 / UP A0A0K1Y874 ** Apis mellifera filamentous virus 1354 residues GB AKY03074, GB YP_009165756 / UP A0A0K1Y866 **

All sequence codes are from UniProt (UP; www.uniprot.org) or GenBank (GB; www.ncbi.nlm.nih.gov/genbank). Sequence searches were performed with MMP CDs within UniProt (www.uniprot.org) or the National Center for Biotechnology Information (blast.ncbi.nlm.nih.gov/Blast.cgi) using standard parameters. Taxonomy according to the International Committee on Taxonomy of Viruses (ictvonline.org/virusTaxonomy.asp), NCBI (www.ncbi.nlm.nih.gov/taxonomy) or the Catalogue of Life (http://www.catalogueoflife.org/col; [1]). # Xestia and Cydia MMPs are the only viral enzymes studied. * These entries have only minimal differences. ** These entries are identical. Searches completed on 10 January, 2017.

Marino-Puertas et al. 5

Suppl. Table 2 — Selected archaeal MMP sequences

Phylum Euryarchaeota Class Halobacteria Haladaptatus sp. 344 residues GB WP_066144928 / UP A0A166SWC5 Halomicrobium mukohataei 392 residues UP C7NZX4 (Haloarcula mukohataei) Class Methanomicrobia Methanocalculus sp. 177 residues UP A0A101H2S1 * Methanococcoides burtonii 231 residues GB WP_011499923 / UP Q12UU6 Methanococcoides methylutens 233 residues UP A0A099T1M7 Methanoculleus sp. 194 residues UP A0A0Q1AIF2 Methanohalobium evestigatum 240 residues GB WP_013195212 / UP D7EBD0 Methanohalophilus mahii 231 residues GB WP_013038296 / UP D5E866 Methanolobus tindarius 241 residues GB WP_023845349 / UP W9DPH7 Methanolobus psychrophilus 235 residues GB WP_015053499 / UP K4MC96 Methanomicrobiales archaeon 177 residues UP A0A117MHP9 * Methanomethylovorans hollandica 242 residues GB WP_015323622 / UP L0KWK3 Methanoregula formicica 179 residues GB WP_015286272 / UP L0HJR6 Methanosalsum zhilinae 227 residues GB WP_013899163 / UP F7XME3 (Methanohalophilus zhilinae) Methanosarcina acetivorans 211 residues UP Q8TIZ9 Methanosarcina barkerii 217 residues UP A0A0E3R7C9 Methanosarcina flavescens 217 residues GB WP_054299817 / UP A0A0P6VKR0 Methanosarcina horonobensis 210 residues UP A0A0E3SB37 Methanosarcina lacustris 215 residues GB WP_048124635 / UP A0A0E3RZU1 Methanosarcina mazei 220 residues GB KKG06863 / UP A0A0F8EZR8 (Methanosarcina frisia) Methanosarcina siciliae 211 residues UP A0A0E3PQS9 Methanosarcina vacuolata 217 residues UP A0A0E3LI77

Phylum Thaumarchaeota Class — / Order Cenarchaeales Cenarchaeum symbiosum 345 residues UP A0RWX1 Class — / Order Nitrosopumilales Candidatus Nitrosoarchaeum 253 residues UP F9CWY2 koreensis Candidatus Nitrosoarchaeum limnia 283 residues GB WP_010190351 / UP S2E6F9 Candidatus Nitrosopumilus 296 residues UP A0A0D5C2T1 adriaticus Candidatus Nitrosopumilus koreensis 238 residues UP K0B4I4 Candidatus Nitrosopumilus salaria 269 residues GB WP_008298808 / UP I3D381 Class — / Order Nitrososphaeria Candidatus Nitrososphaera 251 residues UP A0A075MRP9 evergladensis Unclassified Thaumarcheota Candidatus Nitrosotalea devanaterra 423 residues UP A0A128A209 Thaumarchaeota archaeon 540 residues GB WP_048194249 / UP V6AQP1 Candidatus Nitrosopelagicus brevis 505 residues UP A0A0A7V069

Unclassified Archaea Candidatus Pacearchaeota archaeon 301 residues UP A0A1F6ZFA3

All sequence codes are from UniProt (UP; www.uniprot.org) or GenBank (GB; www.ncbi.nlm.nih.gov/genbank). Sequence similarity searches were performed with karilysin CD (UP D0EM77) within UniProt using standard parameters. Only one representative from each species or genus (when sp.) was chosen. All sequences were manually curated. Marino-Puertas et al. 6

Taxonomy according to the Catalogue of Life (http://www.catalogueoflife.org/col; [1]) or, when absent, UniProt (www.uniprot.org). * Identical sequences. Searches completed on 23 January, 2017.

Marino-Puertas et al. 7

Suppl. Table 3 — Selected bacterial MMP sequences

Phylum Acidobacteria Class Solibacteres Solibacter usitatus 442 residues GB WP_011688765 / UP Q02908

Phylum Actinobacteria Class Acidimicrobiia Acidithrix ferrooxidans 358 residues UP A0A0D8HKH4 Class Actinobacteria Pseudarthrobacter phenanthrenivorans 687 residues GB WP_013599449 / UP F0M7V4 (Arthrobacter phenanthrenivorans) Pseudarthrobacter siccitolerans 686 residues UP A0A024GZ92 Streptomyces lincolnensis 610 residues GB WP_067438548 / UP A0A1B1MG04 Streptomyces scabiei 359 residues UP A0A086GV39 Class - / Order Actinomycetales Alloactinosynnema sp. 556 residues GB WP_054055428 / UP A0A0H5CVK8 Arthrobacter nitrophenolicus 634 residues GB WP_009356397 / UP L8TT82 Knoellia aerolata 271 residues GB WP_052113283 / UP A0A0A0JTC1 Pseudonocardia dioxanivorans 500 residues GB WP_013678450 / UP F4CV22

Phylum Bacteroides Class Bacteroidia Parabacteroides distasonis 446 residues UP A0A174VNZ8 Tannerella forsythia (Bacteroides 472 residues UP D0EM77 forsythus) * Class Cytophagia Cyclobacterium marinum (Flectobacillus 482 residues GB WP_014022474 / UP G0J0B7 marinus)

Phylum Cyanobacteria Class Cyanophyceae Microcystis aeruginosa 268 residues GB WP_052734169 / UP A0A0F6U407 Synechococcus sp. 439 residues UP B4WJ70 Class - / Order Synechococcales Acaryochloris marina 580 residues GB WP_012164578 / UP B0CCT5 Phormidesmis priestleyi Ana 516 residues UP A0A0P7ZK60

Phylum Firmicutes Class Bacilli Bacillus cereus 258 residues GB WP_000775479 / UP R8XLT0 Bacillus mycoides 253 residues UP A0A090ZAN4 Halobacillus halophilus (Sporosarcina 390 residues GB WP_014642607 / UP I0JKN6 halophila) Lactobacillus acidifarinae 228 residues GB WP_057801390 / UP A0A0R1LQY6 Lactobacillus acidophilus 222 residues GB WP_003548046, GB YP_194247 / UP Q5FJA8 Lactobacillus apodemi 193 residues GB WP_035459587 / UP A0A0R1TU85 Lactobacillus brevis 223 residues GB WP_011666964 / UP Q03TZ3 Lactobacillus plantarum 266 residues GB WP_003643352 / UP M4KPC6 Lactobacillus nodensis 240 residues UP A0A0R1KCP7 Lactobacillus senmaizukei 238 residues UP A0A0R2DS50 Marino-Puertas et al. 8

Lactobacillus sunkii 233 residues GB WP_057825587 / UP A0A0R1KWI9 Leuconostoc kimchii 226 residues GB WP_013103335 / UP D5T391 Paenibacillus terrae 280 residues GB WP_044649112 / UP A0A0D7WTR0 Streptococcus suis 231 residues GB WP_044681537 / UP A0A0Z8JY51

Phylum Nitrospirae Class Nitrospira Candidatus Magnetoovum chiemensis 426 residues UP A0A0F2J1S3

Phylum Plantomycetes Class Plantomycetia Candidatus Brocadia fulgida 418 residues UP A0A0M2UXB6 Candidatus Jettenia caeni 420 residues GB WP_007220894 / UP I3IJ81 Candidatus Scalindua brodae 382 residues UP A0A0B0EQJ8 Class Plantomycetacia Isosphaera pallida 435 residues GB WP_013565370 / UP E8QY87 Pirellula staleyi (Pirella staleyi) 281 residues GB WP_012911897 / UP D2R985 Planctomyces sp. 499 residues GB WP_068412701 / UP A0A142YDV0 Singulisphaera acidiphila 538 residues UP L0DHV3

Phylum Proteobacteria Class Alphaproteobacteria Aurantimonas sp. 321 residues GB WP_055845852 / UP A0A0Q6EYY6 Aureimonas sp. 484 residues GB WP_056689492 / UP A0A0Q6EFI8 Bradyrhizobium elkanii 454 residues UP A0A1E3EYM1 Bradyrhizobium jicamae 318 residues GB WP_057837259 / UP A0A0R3LGN7 Bradyrhizobium lablabi 324 residues GB WP_057857189 / UP A0A0R3N9Q1 Hyphomicrobium nitrativorans 291 residues GB WP_023788197 / UP V5SJS8 Labrenzia alexandrii (Stappia alexandrii) 378 residues UP B9QV57 Methylobacterium sp. 470 residues UP B0UC88 Puniceibacterium sp. 271 residues GB WP_053078859 / UP A0A0J5QD38 Rhizobium sp. 404 residues GB WP_062553422 / UP A0A0Q7YFP6 Rhodomicrobium udaipurense 248 residues GB WP_037237908 / UP A0A037UYG6 Rhodospirillaceae bacterium 357 residues UP A0A0F2S3S3 Sphingomonas changbaiensis 556 residues UP A0A0E9MSC9 Sulfitobacter geojensis 501 residues GB WP_064223228 / UP A0A196QSA6 Class Betaproteobacteria Burkholderia gladioli (Phytomonas 463 residues UP A0A0M2QDA5 marginata) Burkholderia vietnamiensis 1065 residues GB WP_059890570 / UP A0A132D6D9 Paraburkholderia glathei 1101 residues UP A0A0J1D520 Rubrivivax gelatinosus 633 residues UP I0HQ73 Class Deltaproteobacteria Chondromyces apiculatus 463 residues UP A0A017SZ72 Chondromyces crocatus 433 residues UP A0A0K1EA09 Labilithrix luteola 314 residues UP A0A0K1Q883 Sandaracinus amylolyticus 588 residues GB WP_053234042 / UP A0A0F6W3W9 Marino-Puertas et al. 9

Sorangium cellulosum (Polyangium 247 residues UP A0A150T1A1 cellulosum) Stigmatella aurantiaca 477 residues GB WP_002612142 / UP Q098Y1 Vulgatibacter incomptus 602 residues GB WP_050725892 / UP A0A0K1PDK5 Class Gammaproteobacteria Acinetobacter sp. 205 residues GB WP_005215616 / UP N9NAE2 Methylophaga aminisulfidivorans 237 residues GB WP_007146704 / UP F5T331 Methylothermaceae bacteria 432 residues UP A0A148N5W8 Pseudoalteromonas luteoviolacea 1466 residues GB WP_065788537 / UP A0A1C0TT69 Candidatus Tenderia electrophaga 261 residues UP A0A0S2TI48 Thiolapillus brandeum 387 residues UP W0TNH9

Unclassified Bacteria Dadabacterium bacterium 302 residues UP A0A0T5ZUN1 Latescibacteria bacterium 337 residues UP A0A0S7XD53 Parcubacteria bacterium 310 residues UP A0A0L0LDW4

All sequence codes are from UniProt (UP; www.uniprot.org) or GenBank (GB; www.ncbi.nlm.nih.gov/genbank). Sequence similarity searches were performed with karilysin CD (UP D0EM77) within UniProt using standard parameters. Only one representative from each species or genus (when sp.) was chosen. All sequences were manually curated. Taxonomy according to the Catalogue of Life (http://www.catalogueoflife.org/col; [1]) or, when absent, UniProt (www.uniprot.org). * T. forsythia karilysin is the only bacterial MMP studied. Searches completed on 27 January, 2017.

Marino-Puertas et al. 10

Supplementary References

[1] Y. Roskov, L. Abucay, T. Orrell, D. Nicolson, N. Bailly, P. Kirk, T. Bourgoin, R.E. de Walt, W. Decock, A. de Wever, E. van Nieukerken, Species 2000 & ITIS Catalogue of Life., Species 2000, Leiden (The Netherlands), 2017.